A comparison of aggregation behavior in aqueous humic acids
© The Royal Society of Chemistry and the Division of Geochemistry of the American Chemical Society 2001
Received: 03 January 2001
Accepted: 29 January 2001
Published: 13 February 2001
The ability of six humic acids (HAs) to form pseudomicellar structures in aqueous solution was evaluated by five techniques: size exclusion chromatography; pyrene fluorescence enhancement; the pyrene I1/I3 ratio; the cloud point of dilute HA solutions; and the fluorescence anisotropy of HAs. Soil HAs were found to aggregate most easily, both on microscopic and macroscopic scales. The formation of amphiphilic structures was chiefly related to HA-solvent interactions: highly solvated HAs aggregated poorly, while a lignite derived material underwent intermolecular, rather than intramolecular, rearrangements. A newly discovered algal HA was found to have minimal aggregative properties.
Humic substances are the breakdown products of plant and animal matter in environmental matrices, especially soil. The major components of these materials – humic acid (HA), fulvic acid (FA), and humin – are defined on the basis of their solubilities in aqueous solution. HA, the subject of the present study, is soluble in solutions of pH > 2, while FA is soluble at all pHs and humin is entirely insoluble. HA is comprised of a suite of polydisperse carbonaceous polymers with molecular weights ranging to tens of kDa. Both molecular size and functional group characteristics are variable, depending on the origin of the material and the environmental factors implicated in its formation. Considerable diversity also exists within a single HA sample. Generally, however, HAs have a significant aromatic content (30–60% of C) and an abundance of carboxy (15–25% of C) and hydroxy groups. Their behavior in aqueous solution has been studied extensively [3–6] and it has been noted that they have significant surface activity.
Based on this observation, the detergent model of HA was developed [7–10] and has been found to account for much of its behavior in aqueous solution. According to this model, HA polymers aggregate spontaneously in water, forming micelle-like structures comparable to those formed by synthetic surfactants. The term "pseudomicelle" is often used to describe these bodies, especially in dilute solution, since they are composed of various HA polymer fragments and are likely to be more structurally diverse than customary micelles.[11, 12] It is thought that the formation of humic pseudomicelles in aqueous solution can proceed by both intramolecular and intermolecular mechanisms. [13–15] In the former, a single polymer chain arranges itself in a manner that exposes its hydrophilic parts (e.g., carboxy groups) to the aqueous surroundings, while isolating its hydrophobic portion in the center of the structure. This type of "aggregation" is not characterized by a critical micelle concentration (CMC). It has been shown that the formation of humic pseudomicelles is promoted by the presence of metal ions, especially multivalent ones.
The secondary structure of aqueous HAs can be studied by a variety of methods. Non-intrusive procedures are preferred, since the arrangements of the polymers in water are both changeable and fragile, precluding techniques that interfere with the integrity of the system. Studies of this kind are of interest because of the ubiquitous nature of humic substances in the environment, and their implication in the transport of pollutants through soil. [17–21]
In view of the variations among HAs from different origins, it is important to compare their behavior in aqueous solution, in order to understand how their environmental interactions may differ. The present communication offers a comparison of 6 HAs, studied by 5 different methods.
Humic acids and reagents
Latahco silt loam humic acid (LSLHA) was obtained from the top 30 cm of a Latahco silt loam soil (Argiaquic Xeric Argialbolls), maintained as pasture for at least 20 years. The soil, which contained in addition to silt, 4.15% organic C, 15.9% clay, 12.1% sand, and 0.39% total N (w/w), was air dried and crushed to pass a 2.0 mm sieve. HA extraction was carried out according to the standard procedure published by the International Humic Substances Society. Ritzville soil humic acid (RSHA) was isolated in the same way from a Ritzville silt loam, obtained from Pacific Northwest National Laboratories (Richland, WA). This soil contained (w/w) 43.3% sand, 43.6% silt, 12.3% clay, and 0.7% organic C. Pilayella littoralis humic acid (PLVHA) was obtained from the Barnett Institute (Northeastern University, Boston, MA), where it was isolated from a brown alga by a procedure described by Ghabbour et al. The other HAs used were reference materials obtained from the International Humic Substances Society (St. Paul, MN) and are described in the product literature.
Various characteristics of the 6 HAs used in this study have been discussed previously.[3, 10–12, 15] Relevant information on these materials, in addition to the isolation procedures reported above, includes:
LSLHA: this is a soil HA, considered to be comprised of flexible polymers containing numerous saturated hydrocarbon links.
LHA: this HA is found in association with lignite deposits and is mined at various sites in North America. It is a condensed aromatic material with little molecular flexibility.
SRHA: this is an aquatic HA with a relatively high functional group density. In this respect it resembles a large fulvic acid.
SHHA: this IHSS standard has a high aliphatic and heteroaliphatic content.
RSHA: this material was obtained from a sandy Ritzville soil (0.7% organic carbon). Little is known about its structure, although it appears to have a relatively high aromatic content.
PLVHLA: this new algal HA was obtained from Pilayella littoralis by the Davies group at Northeastern University, Boston. The typical elemental composition was: 42.6% C, 5.2% H, 5.2% N, 0.8% S, 38.5% O, and 6.1% ash. IR, UV, and PMR spectra showed the material to be similar to compost HA.
Pyrene (98%) was obtained from Sigma and purified by recrystallization from absolute ethanol and sublimation onto a cold finger. MgCl2 (J.T. Baker) and aqueous ammonia (Fisher) were used as received. Tris buffer [tris(hydroxymethyl)amino-methane] was obtained from Sigma and prepared as recommended by Cameron et al. All water used was treated with a 0.22 μm Millipore system to 18 MΩ cm resistivity.
Procedures and instrumentation
HA solutions were prepared by placing the appropriate amount of solid in ca. 5 mL of water, dropwise adding conc. aqueous ammonia until dissolution was complete (one drop was generally sufficient), and then diluting with water as needed. The solution was sonicated at ca. 40°C until no more ammonia odor could be detected. The final pH of the solutions was in the range 6.5–7.0. For pyrene containing HA solutions, a 1.0 × l0-7 M aqueous pyrene solution was used as the diluent. The addition of ammonia had no effect on the humic solutes other than expediting their dissolution.
Fluorescence intensity measurements were taken with a Hitachi F-4500 fluorescence spectrophotometer. Data reported as a function of Mg2+ concentration were obtained by adding successive microliter volumes of a 0.1 M MgCl2 solution to 3 mL of HA solution. Dilution effects were thus negligible. Fluorescence intensities were measured exactly l0 min after each salt addition. Pyrene fluorescence was excited at 240 nm (where HAs are only weakly excited) and measured at 372 nm.
SEC separations were carried out on a SigmaChrom GFC-1300 gel filtration column (13.25 mL; 300 × 7.5 mm), supplied by Supelco (Bellefonte, PA). This contains a crosslinked polysaccharide packing which can tolerate eluent pH values in the range 3–12. Cameron et al. noted that used with a Tris buffer, this type of stationary phase minimizes gel–solute interactions other than size exclusion. A Waters (Milford, MA) 510 HPLC pump operating at a back pressure of < 250 psi, and a Waters 411 absorbance detector set at 280 nm were used. The eluent was a 1 M, pH 9, Tris buffer, and the flow rate was 0.5 mL min-1. HA solutions of 50 ppm in Tris were introduced with a 10 μL injection loop. Before each run, the column was eluted with Tris buffer for ca. 1 h, and after completion of all runs it was flushed and stored with 20% ethanol. The void volume of the system was ca. 3 mL.
Cloud point (CP) determinations were made visually on 50 or 100 ppm HA solutions. The ionic strength requirements were determined by heating the solution to 70°C (above the CP in all cases) and maintaining this temperature. The warm solution was titrated with 0.1 M MgCl2 (with temperature equilibration after each addition) until clouding was first observed. The Mg2+ concentration at this point was taken as the minimum needed. The solution was then cooled, with sonication, until the clouding disappeared, and was reheated at a rate of 1–2 deg min-1 until it reappeared. The temperature at this point was recorded as the CP.
The pyrene I1/I3 ratios were measured with a SLM Aminco 8100 fluorescence spectrophotometer. The values were determined by taking the HA/pyrene emission spectrum at 240 nm excitation, subtracting the HA-blank emission at this excitation (which was minimal), and taking the ratio of the emission peaks at 372 nm (I1) and 383 nm (I3). For measurements involving Mg2+, successive aliquots of a MgCl2 solution in the microliter range (causing negligible dilution) were added. Measurements were taken l0 min after each addition.
For the measurement of the HA fluorescence anisotropy, the ⊤-optics arrangement of the SLM Aminco fluorimeter was employed. The instrument has two emission channels, 180° apart, each comprising a Glan-Taylor polarizer. By adjusting one to pass I|| and the other to pass I⊥ (for an explanation of the process, vide infra), the anisotropy could be determined in a single measurement. The reported values, recorded at 340 nm excitation and 400 nm emission, were an average of 40 determinations.
Results and discussion
The clouding phenomenon of amphiphilic solutes in water is best known for nonionic surfactants, especially those with polyoxyethylene (POE) head groups. It refers to a spontaneous phase separations in micellar solutions of these compounds when the temperature is raised to a value known as the cloud point (CP; a characteristic parameter of the surfactant). Clouding is due to the decrease of the dielectric constant of water with temperature, which diminishes its H-bonding with the POE chains, causing their dehydration. In keeping with this, ionic surfactants do not cloud. It has been shown that HA solutions also undergo clouding, provided the ionic strength of the solution is high enough. This requirement is ascribed to the fact that HAs are, to a variable degree, anionic in aqueous solution and will not dehydrate unless the charges are sufficiently neutralized.
Clouding characteristics of HA solutions
Min. MgCI2 conc./103 M
In this technique, both the exciting radiation and the emission used in the fluorescence measurement are passed through polarizers. The static fluorescence anisotropy, r, of a fluorophore is defined in eqn. (1):
where I|| is the emission intensity measured with the emission polarizer aligned parallel with the excitation polarizer, and I⊥ is the intensity obtained with the emission polarizer turned 90°. The value of r, which can attain a maximum of 0.4, is a measure of the rotational diffusion of the fluorophore in solution. Species that diffuse slowly, either due to their molecular size or to solvent interactions (e.g., H-bonding) generally have higher anisotropies than those diffusing quickly.
Conclusions drawn from the measurements discussed above are by necessity tentative, since the results are without exception open to interpretation. It has been noted that the fluorescence enhancement and I1/I3 results are in good agreement, indicating that LSLHA and RSHA form pseudomicelles easily under the influence of cations in solution. SHHA, LHA and SRHA do so to a lesser degree, while PLVHA does not. The CP behavior suggests that LSLHA also aggregates most easily on a macroscopic scale, while its anisotropy indicates that it is not as thoroughly solvated (at least by glycerol) as some of the other HAs. Cautious extension of this observation to other H-bonding media suggests that the behavior of LSLHA in water is related to its lack of strong solvent interactions. The high CP and high anisotropy of SHHA indicate that this HA is well solvated. LHA has a fairly low CP, showing that macroscopic aggregation (the formation of particles that scatter visible light) proceeds more easily than pseudomicellization. This could mean that association is largely intermolecular in this case, which fits the known condensed aromatic character of LHA. The findings on SRHA are most unequivocal among those for the HAs studied: low fluorescence enhancement, low I1I3, high CP, and high anisotropy all indicate that this aquatic HA is well solvated and has little tendency to aggregate – both in the pseudomicellar and the macroscopic sense. An interesting case is presented by PLVHA, which, being a recently discovered material, has not been extensively studied. The SEC data show a broad size distribution, and both fluorescence enhancement and I1/I3 could mean that minimal pseudomicelle formation takes place. The small anisotropy value suggests relatively low solvation, while the high CP indicates the opposite. This discrepancy may in this instance be related to the different solvents used for the two measurements.
The authors thank the EPA (R82-2832-010) for financial support.
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